BACKGROUND:
Several studies have been performed to understand the tissue repair process as
well as the possible effects of laser therapy in wound healing. OBJECTIVES:
To study the behavior of skin wounds induced in the dorsal region of Wistar rats
(Rattus norvegicus), which were submitted to the low-intensity laser therapy at
3.8 J/cm2 dosage, 15mW potency, during 15 seconds. MATERIAL
AND METHODS: The animals (n=12) were divided into two groups - control and
laser-treated. The latter comprised three applications (immediately after surgery,
48 hours and 7 days after induction of surgical wounds). Ten days after surgery
samples of the wounds were collected and submitted to histopathological and histomorphometric
studies. RESULTS: Neovascularization, fibroblast proliferation and
reduced inflammatory infiltrate in surgical wound submitted to laser therapy were
demonstrated. CONCLUSION: Taken together, the results suggest that
low-intensity laser therapy is an effective method to modulate tissue repair,
thus significantly contributing to a faster and more organized healing process.

Keywords: Laser therapy, low-level;
Rats; Wound healing

INTRODUCTION

Tissue repair is a dynamic state
that encompasses different processes, among which inflammation, cell proliferation
and synthesis of elements which constitute the extracellular matrix, such as collagen,
elastin and reticular fibers.1 Collagen synthesis is a fast and harmonic
process that starts with the interstitial lesion and extends up to the end of
the healing phase, when the remodeling of the tissues occurs.2

The
scarring and tissue repair processes occur after trauma or disease.3
Wound repair and restructuring constitute a complex mechanism, in which several
factors contribute to a variety of scarring types, such as hypertrophy, atrophy
or normotrophy of the injured area. These processes comprise three phases: inflammation,
granulation and formation of the extracellular matrix.4 Normally, in
the wound healing process, after the granulation phase starts, there is a slight
predominance of macrophages and an increase in the number of fibroblasts, with
synthesis of a new extracellular matrix, and remodeling of these tissues occurs
with a contraction of the granulation tissue. During the matrix formation phase,
the fibroblasts produce great amounts of extracellular matrix. Collagen synthesis
occurs on the 21st day after the lesion and the return of the skin to its normal
aspect on the 26th day.5 Once the wound is resolved and involved by
granulation tissue, a significant decrease in macrophages and fibroblasts occurs,
and the maturation of the scar becomes relatively acellular.6

Radiation
and is based on a theory developed by physicist Albert Einstein who, in his article
Zur Quantumtheorie der Strahlung, of 1917, presented the physical principles
of stimulated emission (the laser phenomenon), which is classified as high-power
(with destructive potential) or low-power (without destructive potential).7
This therapy was first used by Mester et al., who used 488 and 515nm argon laser.
Subsequently, the helium-neon (He-Ne) laser was introduced, which emits red light
with a wavelength of 632.8nm, and was replaced by a less expensive and more powerful
device, the diode laser, with a wavelength of 660-950nm.8 Experimental
treatments in patients started in the 1970s, following reports of positive results
obtained by irradiation with low-intensity laser therapy (LILT) of cell cultures
and in animal experiments. The studies performed were insufficient to confirm
the beneficial effects of LILT.9,10 effects appeared,11
but failed due to the great number of interventions and to unsatisfactory quality
of methodology.

Several studies have
been conducted in order to understand the wound healing process, aiming to clarify
the different aspects of the granulation tissue, of tissue epithelization and
neoformation, as well as the possible effects of LILT in the tissue repair process.
This study had the objective of evaluating, by means of a histopathologic and
histomorphometric analysis, the clinical-biological behavior of skin wounds induced
in the dorsal area of Wistar rats (Rattus norvegicus) submitted to LILT. In particular,
the effects of LILT on angiogenesis, fibroblast proliferation and the inflammatory
infiltrate were analyzed.

MATERIAL
AND METHODS

Animals

Twelve male Wistar rats (Rattus norvegicus),
six to eight weeks old, weighing 160 to 220 g, provided by the Animal House of
the Centro de Biologia da Reprodução  CBR [Reproduction Biology
Center] of the Universidade Federal de Juiz de Fora (UFJF/MG), were used.
The rat housing at CBR-UFJF has large dumping containers with wire screens and
two exhausters, besides room heaters. The temperature is kept at around 22ºC,
by natural ventilation in summer and with the help of heaters in winter. The lighting
is mixed  natural light and fluorescent light bulbs, automatically controlled
to get on at 6:00 a.m. and off at 6:00 p.m. The animals were kept in individual
polypropylene cages, equipped with beds of selected wood shavings, baby bottles
with water and troughs for palletized chow, under maintenance conditions which
are in agreement with the criteria of the Colégio Brasileiro de Experimentação
Animal [Brazilian College of Animal Experimentation], and submitted to
daily macroscopic evaluations in order to observe signs indicating secondary infection.

The animals were randomly divided
into two groups, one (I) submitted to surgical skin wounds (n=6), and the other
(II) to surgical skin wounds and low-intensity laser therapy (n=6).

In
the treatment protocol used for this study, group I was kept as control and, in
group II, laser treatment was performed; the whole experiment lasted 10 days.

Experimental models

The
dorsal area of the animals was shaved after anesthesia with intraperitoneal ketamine
(100ml/kg) + xylazine (10mg/kg) and, by means of a punch of approximately 10mm
in diameter, a circular fragment of skin tissue was removed.

The
treatment was carried out using low-intensity laser (Twin Laser) with the following
characteristics: infrared emission laser, pulsatile, arsenium and gallium semiconductors,
wavelength of 870nm, peak power of 70mW, mean exit power of 0.5 to 3.5mW and application
through fiber optics. The application was done by the scanning method in the central
area of the wound, thus allowing its uniform treatment. In the control group,
no kind of treatment was used. In group II, the experimental wounds were submitted
to LILT with the following parameters: 15mW of power, a 3.8J/cm2 dose
for 15s on each one of three applications, the first one immediately after surgery,
the second one 48 hours after the surgical procedure, and the third one seven
days after performing the surgical lesion.

After
10 days from the surgery and after laser application, the animals were sacrificed
with an overdose of anesthesia with intraperitoneal ketamine (100ml/kg) + xylazine
(10mg/kg). The samples from the skin lesions were collected so that part of the
skin adjacent to the wound rims and the entire scar tissue in all its depth were
included.

Histopathology and histomorphometry

All skin lesion samples obtained
were fixed in 10% buffered formalin (pH 7) for at least 24 hours. After fixation,
the samples were gradually dehydrated in increasing concentrations of ethanol
(70% to 100%), cleared in xylene, soaked and embedded in paraffin, according
to routine histological methods. The paraffin-embedded fragments were cut with
an 820 Spence microtome and 6?m thick sections were obtained. The histological
slides were kept in an incubator to dry, and then the sections were stained with
hematoxylin and eosin for histological analysis.

Histomorphometry
was performed using images captured and evaluated by a computerized Axion Vision
(Zeiss, Berlin, Germany) image capture system. Images were captured from four
randomly chosen microscopic fields for each histological slide, using the digital
camera (total enlargement 400x) of an Axiostar Plus microscope (Zeiss, Berlin,
Germany). The images were stored and submitted to a count of inflammatory cells,
evaluation of fibroblast proliferation, analysis of the local angiogenesis and
of the diameter of the ulcerated areas of the wounds at the end of the experiment,
using digital marking.

Statistical
analysis

The statistical analysis
of the several parameters evaluated in the two groups was made by a non-parametric
method, the Mann-Whitney test, with a significance level of p<0.05.

RESULTS

The
clinical observation of skin lesion samples of the animals showed a small amount
of clot on the surface, with clear presence of blood vessels in the deep hypodermis.
These vessels, when accidentally ruptured in the dermis or hypodermis region,
filled the lesions partially or totally with clots.

On
the 10th postoperative day, the skin lesions of group I (control) exhibited an
early-phase tissue repair pattern, with formation of a whitish crust, with slightly
elevated rims and a reddish core due to the accentuated presence of blood irrigation
in that area, indicating the presence of granulation tissue. On the other hand,
the wounds of group II, which had been submitted to low-intensity laser treatment,
showed complete tissue repair, exhibiting scars with evident rims and a central
portion slightly unleveled, but presenting an advanced morphological and functional
recovery of the involved tissues.

The
animals, placed in individual cages under maintenance conditions which are in
agreement with the criteria of the Colégio Brasileiro de Experimentação
Animal, were submitted to daily macroscopic evaluations and presented no signs
indicating secondary infection during the experiment.

In
the analysis of the histological results, two distinct areas were evaluated, one
more superficial, involving epithelial proliferation, and the area of connective
tissue beneath the more superficial portion of the lesion. In the control group,
next to the rim of the surgical wound, a discrete epithelial proliferation was
observed, and virtually all over its extension the presence of tissue exhibiting
a broad ulceration area and fibrinonecrotic material over granulation tissue was
seen (Figure 1). However, in group II, the one submitted to
laser therapy, the histopathological study evidenced material showing undamaged
epidermis covering well-developed granulation tissue (Figure 2),
with connective tissue rich in collagen fibers with a parallel orientation with
regard to the surface of the wound, determining a more organized tissue repair
process.

Graph
1 presents the diameter of the ulcerated areas on the 10th postoperative day,
the treated animals having shown a significant reduction in the diameters of the
ulcerated areas at the end of the experiment, as compared to the control group
(p<0.05). As for vascularization, the treated animals presented a significantly
greater number of blood vessels per microscopic field (Graph 2).

The
histomorphometric analysis also demonstrated that the animals treated with LILT
presented a greater number of fibroblasts per microscopic field (Graph
3), where the identification of fibroblasts, based on morphological criteria,
was made by two different researchers. Moreover, it was observed that LILT significantly
reduced the intensity of the inflammatory infiltrate present in the lesions submitted
to treatment (Graph 4).

DISCUSSION

Low-intensity
laser treatment is a method that is accepted by the Food and Drug Administration
(FDA) as an effective clinical treatment for tissue healing, as it has already
been widely studied.12,13In vitro studies suggest that LILT
favors collagen synthesis,14 increases the motility of keratinocytes,15
releasing growth factors,16 besides transforming fibroblasts into myofibroblasts.17

Several
studies used superficial wounds for evaluating the effects of LILT on wound healing.
Some of them used ulcers of different sizes and depths,12,18,19 and
others developed superficial wound models in animals.20-22 These different
methods produced a variety of results and conclusions about the effects of LILT.
In this study, we observed that the surgical lesions submitted to low-intensity
laser treatment, when compared to the lesions in the control group, showed a better
developed tissue repair process, with greater wound contraction and higher epithelial
migration speed.

Several authors reported
on clinical trials which showed the benefits of LILT in tissue healing, but others
found none of these effects.20,23 The conflicting data from the literature
give rise to plausible doubts about variations in the treatment factors and limitations
in experimental designs, including the comparison between clinically heterogeneous
wounds, the need for control groups and the limitations faced in the investigation
of such contradictory results. LILT is still a controversial method, as can be
seen in numerous publications,8,24,25 mainly because its mechanism
of action is unknown.

The field of
action of laser is very broad and the studies showed its contribution to the tissue
repair process, particularly regarding its influence on the modulation of certain
cell types in the wound healing process. Gómez-Villamandos et al. reported
an increase in wound healing after laser therapy, with increased mitotic activity,
number of fibroblasts, collagen synthesis and neovascularization of the injured
tissues.26 Other authors16,27 observed that the production
of fibroblast growth factors (FGF) and the predominance of fibroblasts in the
culture increased considerably after low-intensity laser irradiation. Furthermore,
Bisht et al. reported the development of granulation tissue and epithelization
of wounds in Wistar rats treated with He-Ne laser.28

In
the present study, we evaluated vascular proliferation and observed a smaller
amount of blood vessels in the surgical lesions of the control group (Figure
3) compared to those of the treated group (Figure 4),
where an increase in vascular proliferation was found in the surgical wound samples,
which was also observed when fibroblast proliferation in both groups was evaluated.
In the control group samples, a smaller number of fibroblasts were observed (Figure
5) compared to those from the treated group, where a significant increase
in fibroblast proliferation was found (Figure 6).

Moreover,
when the amount of inflammatory cells present in the lesions of both groups was
analyzed, a greater number of such cells were observed in the control group samples
(Figure 5) compared to those from the treated group (Figure
6), which exhibited a significant decrease of inflammatory infiltrate.

These findings suggest a faster tissue repair
in the group submitted to LILT, expressed by an increase in cell proliferation
and vascularization, besides an expressive decrease in the number of inflammatory
cells.

CONCLUSION

Taken
together, the results suggest that LILT is effective in tissue wound healing,
as observed in an experimental model of surgical wounds produced in rats. This
type of laser therapy showed positive effects, speeding up tissue proliferation,
increasing local vascularization and forming a more organized granulation tissue.